During the previous funding cycle, we identified a mechanism for how hypoxia and reactive oxygen/nitrogen species (ROS/RNS) regulate blood vessel growth (angiogenesis) and radiotherapy responses, via HIF-1 (hypoxia-inducible factor-1) and VEGF (vascular endothelial growth factor). We refer to this as the HRHV axis. Unregulated tumor angiogenesis is known to be tied to hypoxia, HIF1 and VEGF by our work and that of many others, however, it is unclear how the angiogenic stimulus is maintained over time. In this grant, which includes pre-clinical and clinical objectives, we propose to test the overriding hypothesis that activation of the HRHV axis leads to abnormal vascular adaptation and angiogenic shunt flow and, which in turn maintains tumor hypoxic-pathophysiology through positive feedback loops. In Specific Aim 1 we will examine how aberrant angiogenesis and vascular adaptation form feedback loops with the HRHV axis; hypoxia becomes a consequence of shunt flow and abnormal angioadaptation leads to cycling hypoxia. In Specific Aim 2 we will test whether inhibiting the HRHV axis with a novel SOD mimetic +/- VEGF inhibition will improve oxygen transport by inhibiting the feedback loops to restore a more normal vascular network structure. This will in turn improve tumor response and local control rate following radiotherapy. In order to examine the importance of the HRHV axis in human tumors we will establish biomarkers of ROS/RNS stress and cycling hypoxia in pre-clinical models, using matched hypoxia marker drugs to localize regions of hypoxia in the same tumor when breathing air and 10% oxygen. We expect to see differences in temporal patterns of biomarker expression after breathing 10% oxygen, as compared with controls. In Specific Aim 3 we will examine HRHV axis patterns identified in SA2 to assess prevalence of ROS/RNS damage and cycling hypoxia in several human tumor entities: We will acquire material in the course of a clinical study in which human patients with breast cancer will be given the hypoxia marker drug, EF5. We will also investigate archived tissues from prior studies in which patients with cervix, head and neck and breast cancer were administered the hypoxia marker drug, pimonidazole. The hypoxia marker drugs will serve as a marker standard for comparison with biomarker distributions. We will explore potential prognostic significance of the markers of the HRHV axis in retrospective analyses of early stage breast, non small cell lung cancer and cervix cancer (~100 patients each, with known clinical outcome). We expect that the outcome of this project will solidify the importance of the HRHV axis and that it will set the stage for future human trials where the HRHV axis is targeted as a therapeutic strategy in combination with radiotherapy and / or chemotherapy.